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The incidence of pulmonary and venous thromboembolism is increased during the first trimester of pregnancies after assisted reproductive technology (ART) compared to spontaneous conception. We previously found that haemostatic plasma variables changed but within normal limits during controlled ovarian hyperstimulation (COH) concomitant with a major increase in plasma microvesicles (MVs) and markers indicating cell activation. We now explored the proteome of these MVs. Thirty-one women undergoing ART were blood sampled at down-regulation (DR) of oestrogen and at high level stimulation (HLS) with its 10-100-fold increased oestrogen level. Samples were analysed by liquid chromatography and tandem mass spectrometry to identify and quantify the proteome. We identified 306 proteins in the MVs and 72 had changed significantly at HLS compared to DR and more than 20% of them were associated with haemostasis. Thus, proteins related to both haemostasis and complement activation altered in plasma MVs in parallel with MV activation during COH. This needs to be further explored in the clinical context.

Background: The assisted reproductive technique in vitro fertilization (IVF) is associated with an increased risk of venous thromboembolism (VTE) and pulmonary embolism (PE) during the first trimester., Objectives: To compare the incidence of VTE and PE during the first trimester of IVF pregnancies using fresh or frozen-thawed embryo transfer to that during natural pregnancies., Patient/methods: Nationwide Swedish registry-based cohort study of women who gave birth (n = 902 891) at the age of 15-50 years to their first child from the 1st of January 1992 until the 31st of December 2012. Exposure groups were IVF with fresh respectively frozen-thawed embryo transfer. Incidences of VTE and PE were calculated, and time-varying hazard ratios estimated for all trimesters after fresh respectively frozen-thawed embryo transfer IVF and compared to natural conception., Results and Conclusion: Women giving birth after fresh embryo transfer IVF had a more than eightfold increased incidence of venous thromboembolism (hazard ratio [HR] 8.96, 95% CI 6.33 to 12.67) and pulmonary embolism during the first trimester, (HR 8.69, 95% CI 3.83 to 19.71) compared to women giving birth after natural conception. The incidence of VTE in women giving birth after frozen-thawed embryo transfer was not increased during the first trimester. To conclude, fresh embryo transfer IVF was associated with a significantly increased incidence of VTE and PE during the first trimester. These results suggest that frozen-thawed embryo transfer could be a preferred method of IVF with a minimised maternal risk., (© 2020 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International.)

The Karolinska Institutet Human Embryonic Stem Cell Bank (KI Stem Cell Bank) was established at KI, Stockholm, Sweden, when the first human embryonic stem cell (hESC) line was derived by Professor Hovatta and colleagues in 2002. Since then, the bank has grown to include 60 hESC lines. From the very beginning the aim of the bank has been derivation of hESC lines suitable for clinical use. Step by step progress has been made towards this goal, including removal of xeno components, establishment of chemically defined conditions and Good Manufacturing Practice (GMP) compliancy. Today our bank includes such clinical grade hESC line, KARO1, derived and banked according to GMP guidelines. Many of the hESC lines in the bank have been distributed to the scientific community and are deposited in the Stockholm Medical Biobank available for research on collaborative basis., Competing Interests: Declaration of interests Authors declare no conflict of interest, (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. Here, we report single-cell transcriptomes and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our data identify transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data do not support the existence of germline stem cells in adult human ovaries, thereby reinforcing the dogma of a limited ovarian reserve.

Globally, fertility-related issues affect around 15% of couples. In 20%-30% of cases men are solely responsible, and they contribute in around 50% of all cases. Hence, understanding of in vivo germ-cell specification and exploring different angles of fertility preservation and infertility intervention are considered hot topics nowadays, with special focus on the use of human pluripotent stem cells (hPSCs) as a source of in vitro germ-cell generation. However, the generation of male germ cells from hPSCs can currently be considered challenging, making a judgment on the real perspective of these innovative approaches difficult. Ever since the first spontaneous germ-cell differentiation studies, using human embryonic stem cells, various strategies, including specific co-cultures, gene over-expression, and addition of growth factors, have been applied for human germ-cell derivation. In line with the variety of differentiation methods, the outcomes have ranged from early and migratory primordial germ cells up to post-meiotic spermatids. This variety of culture approaches and cell lines makes comparisons between protocols difficult. Considering the diverse strategies and outcomes, we aim in this mini-review to summarize the literature regarding in vitro derivation of human male germ cells from hPSCs, while keeping a particular focus on the culture methods, growth factors, and cell lines used., Competing Interests: The authors declare no conflicts of interest

Study Question: Does the X chromosome inactivation (XCI) of Klinefelter syndrome (KS)-derived human induced pluripotent stem cells (hiPSCs) correspond to female human pluripotent stem cells (hPSCs) and reflect the KS genotype?, Summary Answer: Our results demonstrate for the first time that KS-derived hiPSCs show similar XCI behavior to female hPSCs in culture and show biological relevance to KS genotype-related clinical features., What Is Known Already: So far, assessment of XCI of KS-derived hiPSCs was based on H3K27me3 staining and X-inactive specific transcript gene expression disregarding the at least three XCI states (XaXi with XIST coating, XaXi lacking XIST coating, and XaXe (partially eroded XCI)) that female hPSCs display in culture., Study Design, Size, Duration: The study used hiPSC lines generated from two azoospermic patients with KS and included two healthy male (HM) and one healthy female donor., Participants/materials, Setting, Methods: In this study, we derived hiPSCs by reprograming fibroblasts with episomal plasmids and applying laminin 521 as culture substrate. hiPSCs were characterized by karyotyping, immunocytochemistry, immunohistochemistry, quantitative PCR, teratoma formation, and embryoid body differentiation. XCI and KS hiPSC relevance were assessed by whole genome transcriptomics analysis and immunocytochemistry plus FISH of KS, HM and female fibroblast, and their hiPSC derivatives., Main Results and the Role of Chance: Applying whole genome transcriptomics analysis, we could identify differentially expressed genes (DEGs) between KS and HM donors with enrichment in gene ontology terms associated with fertility, cardiovascular development, ossification, and brain development, all associated with KS genotype-related clinical features. Furthermore, XCI analysis based on transcriptomics data, RNA FISH, and H3K27me3 staining revealed variable XCI states of KS hiPSCs similar to female hiPSCs, showing either normal (XaXi) or eroded (XaXe) XCI. KS hiPSCs with normal XCI showed nevertheless upregulated X-linked genes involved in nervous system development as well as synaptic transmission, supporting the potential use of KS-derived hiPSCs as an in vitro model for KS., Limitations, Reasons for Caution: Detailed clinical information for patients included in this study was not available. Although a correlation between DEGs and the KS genotype could be observed, the biological relevance of these cells has to be confirmed with further experiments. In addition, karyotype analysis for two hiPSC lines was performed at passage 12 but not repeated at a later passage. Nevertheless, since all XCI experiments for those lines were performed between passage 11 and 15 the authors expect no karyotypic changes for those experiments., Wider Implications of the Findings: As KS patients have variable clinical phenotypes that are influenced by the grade of aneuploidy, mosaicism, origin of the X chromosome, and XCI 'escapee' genes, which vary not only among individuals but also among different tissues within the same individual, differentiated KS hiPSCs could be used for a better understanding of KS pathogenesis., Study Funding/competing Interest(s): This study was supported by grants from the Knut and Alice Wallenberg Foundation (2016.0121 and 2015.0096), Ming Wai Lau Centre for Reparative Medicine (2-343/2016), Ragnar Söderberg Foundation (M67/13), Swedish Research Council (2013-32485-100360-69), the Centre for Innovative Medicine (2-388/2016-40), Kronprinsessan Lovisas Förening För Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation, Jonasson Center at the Royal Institute of Technology, Sweden, and Initial Training Network Marie Curie Program 'Growsperm' (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Study Question: Does first-line chemotherapy affect the quality of ovarian pre-antral follicles and stromal tissue in a population of young patients?, Summary Answer: Exposure to first-line chemotherapy significantly impacts follicle viability, size of residual intact follicles, steroid secretion in culture and quality of the stromal compartment., What Is Known Already: First-line chemotherapy is considered to have a low gonadotoxic potential, and as such, does not represent an indication for fertility preservation. Studies investigating the effects of chemotherapy on the quality of ovarian tissue stored for fertility preservation in young patients are limited and the results sometimes contradictory., Study Design, Size, Duration: We conducted a retrospective cohort study including young patients referred to three centers (Helsinki, Oslo and Tampere) to perform ovarian tissue cryopreservation for fertility preservation between 2003 and 2018., Participants/materials, Setting, Methods: A total of 43 patients (age 1-24 years) were included in the study. A total of 25 were exposed to first-line chemotherapy before cryopreservation, whereas 18 patients were not. Density and size of follicles divided by developmental stages, prevalence of atretic follicles, health of the stromal compartment and functionality of the tissue in culture were evaluated and related to age and chemotherapy exposure. Activation of dormant follicles and DNA damage were also assessed., Main Results and the Role of Chance: Patients exposed to first-line chemotherapy showed a significantly higher density of atretic primordial and intermediary follicles than untreated patients. The intact primordial and intermediary follicles were significantly smaller in size in patients exposed to chemotherapy. Production of steroids in culture was also significantly impaired and a higher content of collagen and DNA damage was observed in the stromal compartment of treated patients. Collectively, these observations may indicate reduced quality and developmental capacity of follicles as a consequence of first-line chemotherapy exposure. Neither increased activation of dormant follicles nor elevated levels of DNA damage in oocyte nuclei were found in patients exposed to chemotherapy., Limitations, Reasons for Caution: The two groups were not homogeneous in terms of age and the patients were exposed to different treatments, which did not allow us to distinguish the effect of specific agents. The limited material availability did not allow us to perform all the analyses on the entire set of patients., Wider Implication of the Findings: This study provides for the first time a comprehensive analysis of the effects of first-line chemotherapy on the health, density and functionality of follicles categorized according to the developmental stage in patients under 24 years of age. When exposed to these treatments, patients were considered at low/medium risk of infertility. Our data suggest a profound impact of these relatively safe therapies on ovarian health and encourages further exploration of this effect in follow-up studies in order to optimize fertility preservation for young cancer patients., Study Funding/competing Interest(s): This study was funded by the Swedish Childhood Cancer Foundation, the Finnish Cancer Society, the Finnish Pediatric Research Foundation, the Väre Foundation for Pediatric Cancer Research, The Swedish Research Council, the Stockholm County Council (ALF project) and Karolinska Institutet. The authors have no conflict of interest to declare., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.)

Pleomorphic adenoma gene 1 (PLAG1) is a transcription factor involved in cancer and growth. We discovered a de novo DNA motif containing a PLAG1 binding site in the promoters of genes activated during zygotic genome activation (ZGA) in human embryos. This motif was located within an Alu element in a region that was conserved in the murine B1 element. We show that maternally provided Plag1 is needed for timely mouse preimplantation embryo development. Heterozygous mouse embryos lacking maternal Plag1 showed disrupted regulation of 1,089 genes, spent significantly longer time in the 2-cell stage, and started expressing Plag1 ectopically from the paternal allele. The de novo PLAG1 motif was enriched in the promoters of the genes whose activation was delayed in the absence of Plag1. Further, these mouse genes showed a significant overlap with genes upregulated during human ZGA that also contain the motif. By gene ontology, the mouse and human ZGA genes with de novo PLAG1 motifs were involved in ribosome biogenesis and protein synthesis. Collectively, our data suggest that PLAG1 affects embryo development in mice and humans through a conserved DNA motif within Alu/B1 elements located in the promoters of a subset of ZGA genes.

In the original article, Fig. 1A was by mistakenly duplicated. The corrected image is provided in this correction article.

Regeneration of injured human heart muscle is limited and an unmet clinical need. There are no methods for the reproducible generation of clinical-quality stem cell-derived cardiovascular progenitors (CVPs). We identified laminin-221 (LN-221) as the most likely expressed cardiac laminin. We produced it as human recombinant protein and showed that LN-221 promotes differentiation of pluripotent human embryonic stem cells (hESCs) toward cardiomyocyte lineage and downregulates pluripotency and teratoma-associated genes. We developed a chemically defined, xeno-free laminin-based differentiation protocol to generate CVPs. We show high reproducibility of the differentiation protocol using time-course bulk RNA sequencing developed from different hESC lines. Single-cell RNA sequencing of CVPs derived from hESC lines supported reproducibility and identified three main progenitor subpopulations. These CVPs were transplanted into myocardial infarction mice, where heart function was measured by echocardiogram and human heart muscle bundle formation was identified histologically. This method may provide clinical-quality cells for use in regenerative cardiology., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

In the past, explant tissue-culture methodologies have been used to grow gonads and study their development. Results from in vitro cultures of human gonads showed limited progress toward gonadal cell differentiation and were focused mainly on germ-cell differentiation. Thus, detailed studies focusing on human first-trimester gonadal tissue functionality in vitro are still missing. In this study we investigated the endocrine function of human first-trimester gonads in vitro. We included 27 female and 28 male gonadal samples, derived from a total of 55 cases, at postconceptional ages of 4.5 to 10.5 weeks. Tissues were cultured using an explant tissue-culture system for 14 days. Assays for testosterone (liquid chromatography-tandem mass spectrometry), anti-Müllerian hormone (AMH; ELISA), and inhibin B (ELISA) were performed using media collected after 7 and 14 days of culture. We demonstrated sex- and age-dependent secretion profiles of testosterone, AMH, and inhibin B in the culture media, which resemble the pattern of hormone production in human gonads in vivo, from the few available studies at the same age range. Our study shows that explant tissue-culture conditions are robust for culture of human first-trimester gonadal somatic cells. Thus, it can be used to study human gonadal development and related diseases as well as the effect of potentially hormone-disturbing substances in human gonads during development. However, detailed molecular studies are needed for better understanding of the mechanistic control of the endocrine function of human first-trimester gonads.

Primary ovarian insufficiency (POI) affects ∼1% of women before 40 years of age. The recent leap in genetic knowledge obtained by next generation sequencing (NGS) together with animal models has further elucidated its molecular pathogenesis, identifying novel genes/pathways. Mutations of >60 genes emphasize high genetic heterogeneity. Genome-wide association studies have revealed a shared genetic background between POI and reproductive aging. NGS will provide a genetic diagnosis leading to genetic/therapeutic counseling: first, defects in meiosis or DNA repair genes may predispose to tumors; and second, specific gene defects may predict the risk of rapid loss of a persistent ovarian reserve, an important determinant in fertility preservation. Indeed, a recent innovative treatment of POI by in vitro activation of dormant follicles proved to be successful., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Human embryonic stem (hES) cells represent an important tool to study early cell development. The previously described use of human recombinant laminin (LN) 521 represented a step forward in generating clinically safe culture conditions. To test the short-term effect of LN521 on cultured hES cells, five male hES cell lines were cultured on human foreskin fibroblasts (hFFs), Matrigel, LN521, and LN121 and characterized by qPCR, immunofluorescence analysis, as well as their potential for three-germ layer differentiation. Variations in gene expression related to pluripotency, stemness, and testicular cells at different passages and culture conditions were evaluated by qPCR. All cell lines expressed pluripotency markers at protein and RNA level and were able to differentiate into cell types of the three germ layers after being cultured on LN521 for nine passages. Reduction in variation of pluripotency marker expression could be observed after culturing the cells on LN521 for nine passages. hES cells cultured on LN521 exhibited less differentiation, faster cell growth, and attachment when compared to hES cells cultured on LN121 or Matrigel. Our results indicate a positive effect of LN521 in stabilizing pluripotency gene expression and might be the first step towards more controllable and robust culture conditions for hES cells.

Infertility is a global health problem with an estimated incidence of 15%. Exposure to chemicals is a potential causal factor, and there is a lack of studies examining the effects on female germ cells. Here, we have studied the impact of different aryl hydrocarbon receptor (AHR) modulators on human ovarian follicles using a human ovarian tissue culture model. Expression of AHR was analyzed in tissue samples, and effects of the selected ligands resveratrol (RSVL), 6-formylindolo(3,2-b)carbazole (FICZ), and alpha-naphthoflavone (aNF) on AHR transactivation studied in a granulosa cell tumor line. Cortical human ovarian tissue containing preantral follicles was exposed to the ligands or vehicle (dimethylsulfoxide, DMSO) for seven days in vitro. Follicle growth was assessed by counting and measuring follicles from serial tissue sections, cell death quantified using in situ Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay, and steroid hormone production measured using a newly developed ultra-performance liquid chromatography method. AHR was expressed in all donated ovarian tissue samples. FICZ induced AHR transactivation in the granulosa cell line while aNF antagonised it. Compared to DMSO control, FICZ had no effect on follicles in culture, RSVL increased the proportion of growing follicles, and aNF increased cell death, disrupted growth of secondary follicles, increased testosterone, and reduced estradiol levels. We conclude that RSVL supports and aNF disrupts growth of human ovarian follicles in culture. We further conclude that the human ovarian tissue culture model is suitable for studying effects of chemicals on follicular biology., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Chloride absorption and bicarbonate excretion through exchange by the solute carrier family 26 member 3 (SLC26A3) and cystic fibrosis transmembrane conductance regulator (CFTR) are crucial for many tissues including sperm and epithelia of the male reproductive tract. Homozygous SLC26A3 mutations cause congenital chloride diarrhea with male subfertility, while homozygous CFTR mutations cause cystic fibrosis with male infertility. Some homozygous or heterozygous CFTR mutations only manifest as male infertility. Accordingly, we studied the influence of SLC26A3 on idiopathic infertility by sequencing exons of SLC26A3 in 283 infertile and 211 control men. A heterozygous mutation c.2062 G > C (p.Asp688His) appeared in nine (3.2%) infertile men, and additionally, in two (0.9%) control men, whose samples revealed a sperm motility defect. The p.Asp688His mutation is localized in the CFTR-interacting STAS domain of SLC26A3 and enriched in Finland, showing a significant association with male infertility in comparison with 6,572 Finnish (P < 0.05) and over 120,000 global alleles (P < 0.0001) (ExAC database). Functional studies showed that while SLC26A3 is a strong activator of CFTR-dependent anion transport, SLC26A3-p.Asp688His mutant retains normal Cl - /HCO 3 - exchange activity but suppresses CFTR, despite unaffected domain binding and expression. These results suggest a novel mechanism for human male infertility─impaired anion transport by the coupled SLC26A3 and CFTR.

Stem cells display a fundamentally different mechanism of proliferation control when compared to somatic cells. Uncovering these mechanisms would maximize the impact in drug discovery with a higher translational applicability. The unbiased approach used in phenotype-based drug discovery (PDD) programs can offer a unique opportunity to identify such novel biological phenomenon. Here, we describe an integrated phenotypic screening approach, employing a combination of in vitro and in vivo PDD models to identify a small molecule increasing stem cell proliferation. We demonstrate that a combination of both in vitro and in vivo screening models improves hit identification and reproducibility of effects across various PDD models. Using cell viability and colony size phenotype measurement we characterize the structure activity relationship of the lead molecule, and identify that the small molecule inhibits phosphorylation of ERK2 and promotes stem cell proliferation. This study demonstrates a PDD approach that employs combinatorial models to identify compounds promoting stem cell proliferation.

Nonpluripotent neural progenitor cells (NPCs) derived from the human fetal central nervous system were found to express a number of messenger RNA (mRNA) species associated with pluripotency, such as NANOG, REX1, and OCT4. The expression was restricted to small subpopulations of NPCs. In contrast to pluripotent stem cells, there was no coexpression of the pluripotency-associated genes studied. Although the expression of these genes rapidly declined during the in vitro differentiation of NPCs, we found no evidence that the discrete expression was associated with the markers of multipotent neural stem cells (CD133 + /CD24 lo ), the capacity of sphere formation, or high cell proliferation rates. The rate of cell death among NPCs expressing pluripotency-associated genes was also similar to that of other NPCs. Live cell imaging showed that NANOG- and REX1-expressing NPCs continuously changed morphology, as did the nonexpressing cells. Depletion experiments showed that after the complete removal of the subpopulations of NANOG- and REX1-expressing NPCs, the expression of these genes appeared in other NPCs within a few days. The percentage of NANOG- and REX1-expressing cells returned to that observed before depletion. Our results are best explained by a model in which there is stochastic transient expression of pluripotency-associated genes in proliferating NPCs.

POLR3G is expressed at high levels in human pluripotent stem cells (hPSCs) and is required for maintenance of stem cell state through mechanisms not known in detail. To explore how POLR3G regulates stem cell state, we carried out deep-sequencing analysis of polyA + and smallRNA transcriptomes present in hPSCs and regulated in POLR3G-dependent manner. Our data reveal that POLR3G regulates a specific subset of the hPSC transcriptome, including multiple transcript types, such as protein-coding genes, long intervening non-coding RNAs, microRNAs and small nucleolar RNAs, and affects RNA splicing. The primary function of POLR3G is in the maintenance rather than repression of transcription. The majority of POLR3G polyA + transcriptome is regulated during differentiation, and the key pluripotency factors bind to the promoters of at least 30% of the POLR3G-regulated transcripts. Among the direct targets of POLR3G, POLG is potentially important in sustaining stem cell status in a POLR3G-dependent manner., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

For quality assurance (QA) in stem cell banking, a planned system is needed to ensure that the banked products, stem cells, meet the standards required for research, clinical use, and commercial biotechnological applications. QA is process oriented, avoids, or minimizes unacceptable product defects, and particularly encompasses the management and operational systems of the bank, as well as the ethical and legal frameworks. Quality control (QC ) is product oriented and therefore ensures the stem cells of a bank are what they are expected to be. Testing is for controlling, not assuring, product quality, and is therefore a part of QC , not QA. Like QA, QC is essential for banking cells for quality research and translational application (Schwartz et al., Lancet 379:713-720, 2012). Human embryonic stem cells (hESCs), as cells derived from donated supernumerary embryos from in vitro fertilization (IVF) therapy, are different from other stem cell types in resulting from an embryo that has had two donors . This imposes important ethical and legal constraints on the utility of the cells, which, together with quite specific culture conditions, require special attention in the QA system. Importantly, although the origin and derivation of induced pluripotent stem cells (iPSCs ) differ from that of hESCs, many of the principles of QA for hESC banking are applicable to iPSC banking (Stacey et al., Cell Stem Cell 13:385-388, 2013). Furthermore, despite differences between the legal and regulatory frameworks for hESC and iPSC banking between different countries, the requirements for QA are being harmonized (Stacey et al., Cell Stem Cell 13:385-388, 2013; International Stem Cell Banking Initiative, Stem Cell Rev 5:301-314, 2009).

The mechanisms underlying human germ cell development are largely unknown, partly due to the scarcity of primordial germ cells and the inaccessibility of the human germline to genetic analysis. Human embryonic stem cells can differentiate to germ cells in vitro and can be genetically modified to study the genetic requirements for germ cell development. Here, we studied NANOS3 and DAZL, which have critical roles in germ cell development in several species, via their over expression in human embryonic stem cells using global transcriptional analysis, in vitro germ cell differentiation, and in vivo germ cell formation assay by xenotransplantation. We found that NANOS3 over expression prolonged pluripotency and delayed differentiation. In addition, we observed a possible connection of NANOS3 with inhibition of apoptosis. For DAZL, our results suggest a post-transcriptional regulation mechanism in hES cells. In addition, we found that DAZL suppressed the translation of OCT4, and affected the transcription of several genes associated with germ cells, cell cycle arrest, and cell migration. Furthermore, DAZL over expressed cells formed spermatogonia-like colonies in a rare instance upon xenotransplantation. These data can be used to further elucidate the role of NANOS3 and DAZL in germ cell development both in vitro and in vivo., Competing Interests: The authors have declared that no competing interests exist.

A major hurdle for in vitro culturing of primary endothelial cells (ECs) is that they readily dedifferentiate, hampering their use for therapeutic applications. Human embryonic stem cells (hESCs) may provide an unlimited cell source; however, most current protocols deriving endothelial progenitor cells (EPCs) from hESCs use direct differentiation approaches albeit on undefined matrices, yet final yields are insufficient. We developed a method to culture monolayer hESCs on stem cell niche laminin (LN) LN511 or LN521 matrix. Here, we report a chemically defined, xeno-free protocol for differentiation of hESCs to EPCs using LN521 as the main culture substrate. We were able to generate ∼95% functional EPCs defined as VEGFR2 + CD34 + CD31 + VE-Cadherin + . RNA-sequencing analyses of hESCs, EPCs, and primary human umbilical vein endothelial cells showed differentiation-related EC expression signatures, regarding basement membrane composition, cell-matrix interactions, and changes in endothelial lineage markers. Our results may facilitate production of stable ECs for the treatment of vascular diseases and in vitro cell modeling., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Leucine twenty homeobox (LEUTX) is a paired (PRD)-like homeobox gene that is expressed almost exclusively in human embryos during preimplantation development. We previously identified a novel transcription start site for the predicted human LEUTX gene based on the transcriptional analysis of human preimplantation embryos. The novel variant encodes a protein with a complete homeodomain. Here, we provide a detailed description of the molecular cloning of the complete homeodomain-containing LEUTX Using a human embryonic stem cell overexpression model we show that the complete homeodomain isoform is functional and sufficient to activate the transcription of a large proportion of the genes that are upregulated in human embryo genome activation (EGA), whereas the previously predicted partial homeodomain isoform is largely inactive. Another PRD-like transcription factor, DPRX, is then upregulated as a powerful repressor of transcription. We propose a two-stage model of human EGA in which LEUTX acts as a transcriptional activator at the 4-cell stage, and DPRX as a balancing repressor at the 8-cell stage. We conclude that LEUTX is a candidate regulator of human EGA., Competing Interests: The authors declare no competing or financial interests., (© 2016. Published by The Company of Biologists Ltd.)

The hydroxysteroid (17beta) dehydrogenase (HSD17B)12 gene belongs to the hydroxysteroid (17β) dehydrogenase superfamily, and it has been implicated in the conversion of estrone to estradiol as well as in the synthesis of arachidonic acid (AA). AA is a precursor of prostaglandins, which are involved in the regulation of female reproduction, prompting us to study the role of HSD17B12 enzyme in the ovarian function. We found a broad expression of HSD17B12 enzyme in both human and mouse ovaries. The enzyme was localized in the theca interna, corpus luteum, granulosa cells, oocytes, and surface epithelium. Interestingly, haploinsufficiency of the HSD17B12 gene in female mice resulted in subfertility, indicating an important role for HSD17B12 enzyme in the ovarian function. In line with significantly increased length of the diestrous phase, the HSD17B +/- females gave birth less frequently than wild-type females, and the litter size of HSD17B12 +/- females was significantly reduced. Interestingly, we observed meiotic spindle formation in immature follicles, suggesting defective meiotic arrest in HSD17B12 +/- ovaries. The finding was further supported by transcriptome analysis showing differential expression of several genes related to the meiosis. In addition, polyovular follicles and oocytes trapped inside the corpus luteum were observed, indicating a failure in the oogenesis and ovulation, respectively. Intraovarian concentrations of steroid hormones were normal in HSD17B12 +/- females, whereas the levels of AA and its metabolites (6-keto prostaglandin F1alpha, prostaglandin D 2 , prostaglandin E 2 , prostaglandin F 2α , and thromboxane B 2 ) were decreased. In conclusion, our study demonstrates that HSD17B12 enzyme plays an important role in female fertility through its role in AA metabolism.

Cell-derived microparticles (MPs) are known to be elevated in a number of diseases related to arterial and venous thromboembolism (VTE), such as acute myocardial infarction, VTE (deep-vein thrombosis and pulmonary embolism) and peripheral arterial disease. IVF-associated pregnancies have previously been shown to be associated with an increased incidence of VTE, mechanisms behind being unknown and sparsely studied. Our objective was to assess cell activation during IVF through analysis of MP levels and phenotype following ovarian stimulation. Thirty-one women undergoing IVF were included and blood samples were collected at down regulation of oestrogen and at high level stimulation with 10- to 100-fold increased endogenous oestrogen levels. MPs were analysed by flow cytometry and phenotyped according to size and protein expression. We found that overall phosphatidylserine positive platelet-, endothelial- and monocyte-derived MPs significantly increased following ovarian stimulation with increased levels of platelet activation markers CD40 ligand and P-selectin. Furthermore, there was an increase in endothelial-derived MPs exposing activation marker E-selectin and monocyte-derived MPs, while neutrophil-derived MPs decreased slightly. In conclusion we found a major increase in MPs and markers indicating cell activation in parallel with the profound oestrogen boost during IVF. To assess whether these changes in MPs are associated with thromboembolic events requires extended longitudinal studies.

PAIRED (PRD)-like homeobox genes belong to a class of predicted transcription factor genes. Several of these PRD-like homeobox genes have been predicted in silico from genomic sequence but until recently had no evidence of transcript expression. We found recently that nine PRD-like homeobox genes, ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB, NOBOX, TPRX1 and TPRX2, were expressed in human preimplantation embryos. In the current study we characterized these PRD-like homeobox genes in depth and studied their functions as transcription factors. We cloned multiple transcript variants from human embryos and showed that the expression of these genes is specific to embryos and pluripotent stem cells. Overexpression of the genes in human embryonic stem cells confirmed their roles as transcription factors as either activators (CPHX1, CPHX2, ARGFX) or repressors (DPRX, DUXA, TPRX2) with distinct targets that could be explained by the amino acid sequence in homeodomain. Some PRD-like homeodomain transcription factors had high concordance of target genes and showed enrichment for both developmentally important gene sets and a 36 bp DNA recognition motif implicated in Embryo Genome Activation (EGA). Our data implicate a role for these previously uncharacterized PRD-like homeodomain proteins in the regulation of human embryo genome activation and preimplantation embryo development.

Human pluripotent stem cells (hPSCs) hold great promise for applications in regenerative medicine and pharmaceutical development. Such applications require cell culture methods and reagents that are chemically defined, xeno-free, scalable, and low-cost. Herein, we describe non-mechanical passaging of hPSCs on spider silk films under chemically defined and xeno-free conditions. The cells were dissociated into single cells or small aggregates using Accutase or enzyme-free dissociation buffer and then passaged to spider silk films, where they expanded in monolayers until they covered the surface. Cells cultured over 10 passages on spider silk film remained karyotypically normal and pluripotent. In conclusion, a novel method for passaging dissociated hPSCs under conditions that are compatible with clinical applications is presented. The method is cost-efficient and may be useful for both research and clinical applications.

After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts., (Copyright © 2016 John Wiley & Sons, Inc.)

The establishment of permanent human embryonic stem cell lines (hESCs) was first reported in 1998. Due to their pluripotent nature and ability to differentiate to all cell types in the body, they have been considered as a cell source for regenerative medicine. Since then, intensive studies have been carried out regarding factors regulating pluripotency and differentiation. hESCs are obtained from supernumerary human IVF (in vitro fertilization) embryos that cannot be used for the couple's infertility treatment. Today, we can establish and expand these cells in animal substance-free conditions, even from single cells biopsied from eight-cell stage embryos. There are satisfactory tests for the demonstration of genetic stability, absence of tumorigenic mutations, functionality, and safety of hESCs. Clinical trials are ongoing for age-related macular degeneration (AMD) and spinal cord injury (SCI). This review focuses on the present state of these techniques., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Auto-transplant of cryopreserved ovarian tissue in leukemia patients carries a risk to reintroduce malignant cells. Maturation of ovarian follicles in vitro is a promising strategy to overcome the leukemic cell contamination. The follicle development and survival in 14 cryopreserved ovarian tissues with leukemia-specific PCR marker was evaluated after 7 or 14 days culture. Minimal residual disease (MRD) quantification was assessed by real-time quantitative PCR in order to identify the MRD positive (n = 6) and negative (n = 8) samples and to monitor levels of MRD before and after culture. The morphology of ovarian follicles were studied by light microscopy. After culture, no statistical significant differences were detected in follicle densities between MRD positive- and negative samples. Ovarian MRD either decreased below undetectable or fluctuated near the baseline level after 7 and 14 days in culture. This study provides quantitative in vitro evidence that leukemia contamination does not affect the follicle survival in cryopreserved ovarian tissue.

Human embryonic stem cell differentiation towards various cell types belonging to ecto-, endo- and mesodermal cell lineages has been demonstrated, with high efficiency rates using standardized differentiation protocols. However, germ cell differentiation from human embryonic stem cells has been very inefficient so far. Even though the influence of various growth factors has been evaluated, the gene expression of different cell lines in relation to their differentiation potential has not yet been extensively examined. In this study, the potential of three male human embryonic stem cell lines to differentiate towards male gonadal cells was explored by analysing their gene expression profiles. The human embryonic stem cell lines were cultured for 14 days as monolayers on supporting human foreskin fibroblasts or as spheres in suspension, and were differentiated using BMP7, or spontaneous differentiation by omitting exogenous FGF2. TLDA analysis revealed that in the undifferentiated state, these cell lines have diverse mRNA profiles and exhibit significantly different potentials for differentiation towards the cell types present in the male gonads. This potential was associated with important factors directing the fate of the male primordial germ cells in vivo to form gonocytes, such as SOX17 or genes involved in the NODAL/ACTIVIN pathway, for example. Stimulation with BMP7 in suspension culture resulted in up-regulation of cytoplasmic SOX9 protein expression in all three lines. The observation that human embryonic stem cells differentiate towards germ and somatic cells after spontaneous and BMP7-induced stimulation in suspension emphasizes the important role of somatic cells in germ cell differentiation in vitro.

Transcriptional program that drives human preimplantation development is largely unknown. Here, by using single-cell RNA sequencing of 348 oocytes, zygotes and single blastomeres from 2- to 3-day-old embryos, we provide a detailed analysis of the human preimplantation transcriptome. By quantifying transcript far 5'-ends (TFEs), we include in our analysis transcripts that derive from alternative promoters. We show that 32 and 129 genes are transcribed during the transition from oocyte to four-cell stage and from four- to eight-cell stage, respectively. A number of identified transcripts originates from previously unannotated genes that include the PRD-like homeobox genes ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB and LEUTX. Employing de novo promoter motif extraction on sequences surrounding TFEs, we identify significantly enriched gene regulatory motifs that often overlap with Alu elements. Our high-resolution analysis of the human transcriptome during preimplantation development may have important implications on future studies of human pluripotent stem cells and cell reprograming.

Background: Cryopreservation of ovarian tissue has been widely accepted as an option for fertility preservation among cancer patients. Some patients are exposed to chemotherapy prior to ovarian tissue cryopreservation. Consequently, assessment of the developmental capacity of human ovarian tissue after chemotherapy is of primary importance., Materials: In order to study the impact of previous chemotherapy on in vitro development and viability of ovarian follicles, quality control samples from 34 female cancer patients at median age of 15 years (range 1‒35), cryopreserved for fertility preservation before (n = 14) or after (n = 20) initiation of chemotherapy, were thawed and cultured for 7 days. The morphology and developmental stages of ovarian follicles were studied by light microscopy before and after culture. Possible associations between follicular densities, age and exposure to alkylating agents, expressed as cyclophosphamide equivalent dose (CED) were tested., Results: Exposure to chemotherapy significantly impaired the survival and development of ovarian follicles in culture. After seven days, significantly higher densities of intermediary, primary and secondary follicles and lower densities of atretic follicles was detected in the samples collected before chemotherapy. Increasing dose of alkylating agents was identified by multivariate linear regression analysis as an independent predictor of a higher density of atretic follicles, whereas increasing age of the patient predicted a better outcome with less follicle atresia and a higher density of maturing follicles., Conclusion: This study provides quantitative in vitro evidence of the impact of chemotherapy on developmental capacity of cryopreserved human ovarian tissue. The results indicate that fertility preservation should be carried out, if possible, before initiation of alkylating agents in order to guarantee better in vitro survival of ovarian follicles. In addition, ovarian samples from younger girls show lower viability and fewer developing follicles in culture.

Mesenchymal stem cells (MSCs) have vast potential in cell therapy, and are experimentally used in the clinic. Therefore, it is critical to find a serum- and xeno-free cryopreservation method. The aim of this study was to compare two serum- and xeno-free cryoprotectants for MSCs. Adipose tissue MSCs (Ad-MSCs) and bone marrow MSCs (BM-MSCs) were cryopreserved in two cryoprotectants: the defined serum- and xeno-free STEM-CELLBANKER™ (CB) and 10 % dimethyl sulfoxide (DMSO) in a xeno-free serum replacement cell culture medium and compared to non-cryopreserved MSCs. MSCs cryopreserved in CB or DMSO had similar morphology and surface marker expression compared to their respective non-cryopreserved MSC. Ad-MSCs and BM-MSC in both cryoprotectant media exhibited reduced mean fluorescence intensity (MFI) for CD105, BM-MSCs for CD73 and Ad-MSC increased MFI for HLA class I compared to non-cryopreserved MSCs. Population doubling time of CB cryopreserved and non-cryopreserved Ad-MSCs was similar (38.1 ± 13.6 and 36.8 ± 12.1 h), but somewhat higher when cryopreserved in DMSO (42.2 ± 10.8 h). BM-MSCs had higher population doubling time (CB 47.7 ± 11.4 and DMSO 62.3 ± 32.9 h respectively, p < 0.05) compared to Ad-MSCs. The viability of Ad-MSCs was significantly higher after cryopreservation in CB compared to DMSO (90.4 ± 4.5 % vs. 79.9 ± 3.8 % respectively). Ad-MSCs and BM-MSCs retained their mesodermal differentiation potential when cryopreserved in both cryoprotectants. The characteristics of Ad-MSCs post-thawing are better preserved by CB than by DMSO in serum- and xeno-free medium. Furthermore, Ad-MSCs and BM-MSCs are differently affected by the cryoprotectants, which may have implications for cell therapy.

Congenital heart defects (CHD) occur in approximately 50% of patients with Down syndrome (DS); the mechanisms for this occurrence however remain unknown. In order to understand how these defects evolve in early development in DS, we focused on the earliest stages of cardiogenesis to ascertain perturbations in development leading to CHD. Using a trisomy 21 (T21) sibling human embryonic stem cell (hESC) model of DS, we show that T21-hESC display many significant differences in expression of genes and cell populations associated with mesodermal, and more notably, secondary heart field (SHF) development, in particular a reduced number of ISL1(+) progenitor cells. Furthermore, we provide evidence for two candidate genes located on chromosome 21, ETS2 and ERG, whose overexpression during cardiac commitment likely account for the disruption of SHF development, as revealed by downregulation or overexpression experiments. Additionally, we uncover an abnormal electrophysiological phenotype in functional T21 cardiomyocytes, a result further supported by mRNA expression data acquired using RNA-Seq. These data, in combination, revealed a cardiomyocyte-specific phenotype in T21 cardiomyocytes, likely due to the overexpression of genes such as RYR2, NCX, and L-type Ca(2+) channel. These results contribute to the understanding of the mechanisms involved in the development of CHD. Stem Cells 2015;33:1434-1446., (© 2015 AlphaMed Press.)

Small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence. Recent next generation sequencing (NGS) studies have reported the presence of moRNAs in human neurons and cancer cells and in several tissues in mouse, including pluripotent stem cells. In order to gain additional knowledge about human moRNAs and their putative development-related expression, we applied NGS of small RNAs in human embryonic stem cells (hESCs) and fibroblasts. We found that certain moRNA isoforms are notably expressed in hESCs from loci coding for stem cell-selective or cancer-related miRNA clusters. In contrast, we observed only sparse moRNAs in fibroblasts. Consistent with earlier findings, most of the observed moRNAs derived from conserved loci and their expression did not appear to correlate with the expression of the adjacent miRNAs. We provide here the first report of moRNAs in hESCs, and their expression profile in comparison to fibroblasts. Moreover, we expand the repertoire of hESC miRNAs. These findings provide an expansion on the known repertoire of small non-coding RNA contents in hESCs.